Full genome sequence analysis of a 1-7-4-like PRRSV strain in Fujian Province, China

PRRS virus (PRRSV) has undergone rapid evolution and resulted in immense economic losses worldwide. In the present study, a PRRSV strain named FJ0908 causing high abortion rate (25%) and mortality (40%) was detected in a swine herd in China. To determine if a new PRRSV genotype had emerged, we characterized the genetic characteristics of FJ0908. Phylogenetic analysis indicated that FJ0908 was related to 1-7-4-like strains circulating in the United States since 2014. Furthermore, the ORF5 sequence restriction fragment length polymorphism (RFLP) pattern of FJ0908 was 1-7-4. Additionally, FJ0908 had a 100 aa deletion (aa329–428) within nsp2, as compared to VR-2332, and the deletion pattern was consistent with most of 1-7-4 PRRSVs. Collectively, the data of this study contribute to the understanding of 1-7-4-like PRRSV molecular epidemiology in China.


INTRODUCTION
Porcine reproductive and respiratory syndrome (PRRS) is a global viral swine disease and causing severe economic loss in the global pig industry (Neumann et al., 2005;Zhou & Yang, 2010;Holtkamp et al., 2013;Gao et al., 2017). PRRS virus (PRRSV), the causative agent of PRRS, is a small enveloped virus with positive-sense single-stranded RNA virus belonging to the Arteriviridae family in the Nidovirales order (Benfield et al., 1992;Meulenberg, 2000).

Clinical case
In September 2018, severe reproductive and respiratory disease was observed in piglets in a farm. The affected pigs had respiratory distress, and high abortion rate (25%) and mortality (40%).

Strain identification and nucleotide sequencing
PRRSV infection was confirmed by Real-time RT-PCR testing of the serum of affected pigs according to the manufacturer's instructions. The ORF5 sequence RFLP pattern was inferred according to Wesley et al. The complete genomic sequences of FJ0908 were amplified as described previously (Zhang et al., 2018). The PCR products were purified and cloned into pGEM-T Easy according to the manufacturer's instructions (Promega, Madison, WI, USA) and three recombinant clones of every fragment were sequenced by Ruibo Life Technologies Corporation (Beijing, China).

Complete genomic sequence analysis
Fifty-four representative type 2PRRSV sequences in GenBank were utilized in phylogenetic analyses (Table 1). Multiplex sequence alignments were performed using CLUSTAL X (version 1.83) and the phylogenetic relationships were assessed by MEGA 6.0 as described (Liu et al., 2017b). The ORF5 sequences were classified according to the global PRRSV classification systems (Shi et al., 2010).
Recombination events were detected using Simplot v 3.5.1 and the boot scanning analysis was performed with a 200-bp window, sliding along the genome alignments with a step size of 20 bp.

Amino acid analysis of Nsp2
Nsp2 contains different deletions and insertions, as compared to VR2332 and is the most variable protein in PRRSV genome Liu et al., 2017b;Li et al., 2011). Strikingly, the nsp2 gene of the FJ0908 was 2,640 nt in length and encoded 880 aa, with a 100 aa deletion (aa329-428) within nsp2, as compared to VR2332, and the deletion pattern was consistent with most of 1-7-4 PRRSVs.

Phylogenetic analysis
PRRSV ORF5 is the most variable and has been used as a marker of PRRSV genetic variability. Based on global PRRSV classification systems, type 2 PRRSV was divided into nine monophyletic lineages (1-9) and lineage1 was further classified into nine sublineages (1.1-1.9) (Shi et al., 2010;Guo et al., 2018). The ORF5-based phylogenetic tree showed that FJ0908, as well as 1-7-4 isolates including LNWK130, were clustered in sublineage 1.5

Figure 1 Phylogenetic tree based on the ORF5 genes (A) and full length (B) of the FJ0908 and reference viruses.
Reliability of the tree was assessed by bootstrap analysis of 1,000 replications. Our representative isolate FJ0908 were marked with the red triangle ( ). Lineage 1 PRRSVs are divided into nine sublineages.

DISCUSSION
PRRSV causes major economic losses in swine industry since 1990s. Notably, PRRSV continues to expand its genetic diversity. According to Shi et al. (2010), type 2 PRRSV was classified into nine monophyletic lineages based on ORF5 and extensive genetic variation exists among strains within each lineage. It is hard to define the PRRSV homologous, heterologous virus and pathogenic biotype only focused on single gene analysis. To classify and infer the likely pathogenic biotype, RFLP patterns of ORF5 for type 2 strains is standard approach for veterinarians (Wesley et al., 1998). The RFLP pattern 1-7-4 emerged in the US and has become prevalent since 2014, this nomenclature has been associated with severe disease in herds leading to significant economic losses (Van Geelen et al., 2018). In the present study, FJ0908 was isolated in a farm with high abortion rate and mortality in sows, the restriction sites analysis revealed that the ORF5 RFLP of FJ0908 has the 1-7-4 pattern. Comparison to PRRS sequences in GenBank indicated FJ0908 belonged to 1-7-4-like PRRSV. The genomic regions with the highest variation were found in Nsp1β, Nsp2, ORF2, ORF3, ORF4, ORF5a and ORF5, the lowest variation were found in Nsp1α, Nsp8-12, and ORF6 (Table 2). FJ0908 had 100 aa deletions within Nsp2 (corresponding to position 328-427 of VR2332 nsp2), as compared to the reference strain VR2332, and the deletion pattern was consistent with 1-7-4 viruses.
Recombination may play an important mechanism in generating genetic diversity in PRRSV (Liu et al., 2011;Murtaugh et al., 2010). Most of the 1-7-4 PRRSV isolates may be most potentially derived from different recombination patterns occurring among the local strains in the United States (Van Geelen et al., 2018). Recently, 1-7-4-like PRRSV strains, LNWK130 isolated in Liaoning Province, China was reported to originate from recombination events between IA/2014/NADC34 and ISU30. Currently, recombination events involving NADC30-like PRRSV strains and other PRRSV strains frequently occurred in China (Zhao et al., 2015;Zhang et al., 2016;Bian et al., 2017;Liu et al., 2017a;Liu et al., 2017c;Zhao et al., 2017;Wang et al., 2018;Zhou et al., 2018;Liu et al., 2019). To test for possible recombinant events within FJ0908 strain, we performed recombinant detection using SimPlot v3.5.1 software. Recombination analysis performed with the available full-length genome sequences revealed FJ0908 maybe originate from recombination events between IA/2014/NADC34 and ISU30. Although FJ0908 and LNWK130 maybe drive from recombination events between IA/2014/NADC34 and ISU30, the recombination pattern of two strains were different. Two recombination breakpoints were identified in nsp1 (nt 760 and nt 1,300) in FJ0908 strain, whereas one recombination breakpoint in nsp2 (nt 1480) in LNWK130, suggesting the ancestor of FJ0908 and LNWK130 were most probably transported from different region of United States.
In conclusion, 1-7-4-like PRRSV was also detected in Fujian Province of China besides Liaoning Province. Therefore, effective strategy should be taken to control 1-7-4-like PRRSV and to monitor herd movements.

CONCLUSION
In summary, we thoroughly analyzed a new sublineage of PRRSV strain FJ0908 isolated from Fujian Province, China on the basis of a comprehensive study with the full-length genome. These novel sublineage 1.5 virus is closely related to the ORF5 RFLP 1-7-4 strains. Phylogenetic and molecular evolutionary analyses indicated that FJ0908 originated from a natural recombination event between IA/2014/NADC34 and ISU30. Our data enhance our understanding of the PRRSV evolution in China.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This study was supported by the Leading Project Foundation of Science Department of Fujian Province (2018N0022), the Major Project of Science and Technology Program of Fujian Province, China (2019NZ09005) and the Fujian Natural Science Foundation, Fujian Province, China (2016J01168). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors: